This invention relates to methods for measuring bacterial growth and antibiotic resistance, and particularly to such measurements using a Suspended Microchannel Resonator (SMR) and measuring the mass of multiple bacteria over time and in changing fluidic environments.
Precision measurements of nanometer- and micrometer-scale particles, including living cells and multicellular entities such as bacteria, have wide application in pharmaceuticals/drug delivery and disease studies, as well as in other major industries and fields of research. This need is growing due to the need to better understand and treat diseases and develop and maintain effective treatments and drugs.
A variety of particle sizing and counting techniques, such as light scattering, Coulter Counters and others are known in the art. These techniques are embodied in commercial instruments and are used in industrial, medical, and research applications. Although such techniques have proven utility, they have limitations that limit their applicability. Relatively recently, particle detection and measurement based on the use of SMR's has been developed, and shows promise of going beyond some of the limitations of conventional techniques. The SMR uses a fluidic microchannel embedded in a resonant structure, typically in the form of a cantilever or torsional structure. Fluids, possibly containing target particles, are flowed through the sensor, and the contribution of the particles to the total mass within the sensor causes the resonance frequency of the sensor to change in a measurable fashion. SMR's are typically microfabricated MEMS devices. The use of microfabricated resonant mass sensors to measure fluid density has been known in the literature for some time [P. Enoksson, G. Stemme, E. Stemme, “Silicon tube structures for a fluid-density sensor”, Sensors and Actuators A 54 (1996) 558-562]. However, the practical use of resonant mass sensors to measure properties of individual particles and other entities suspended in fluid is relatively recent, as earlier fluid density sensors were not designed to measure individual particles at the micron and submicron scale.
In a body of work including work by the inventors of this application, miniaturization and improvement of several orders of magnitude in mass resolution has been demonstrated. Development in the microfabrication recipes, the fluidics design, and measurement techniques are described in a number of co-pending patent applications and scientific publications. In particular U.S. patent application Ser. Nos. 11/620,320, 12/087,495, and 12/305,733 are particularly relevant and are incorporated by reference in their entirety. Also of relevance is a publication by others including the current inventors, [T. P. Burg, M. Godin, S. M. Knudsen et al., “Weighing of biomolecules, single cells and single nanoparticles in fluid,” Nature 446 (7139), 1066-1069 (2007)] By using the microfabrication techniques described in the references, SMR sensors have been fabricated with mass resolution of less than 1 femtogram (10−15 g). This resolution is sufficient to detect and measure the mass of individual particles in the range of −100 nanometers up to many microns in size, including living cells.
Improvements in SMR based measurement techniques have been disclosed, which allow for a particle to be held in the measurement portion of the SMR for extended periods of time. Although the disclosed techniques have the advantage of improving signal to noise, they also provide for the ability to measure particle properties which may change over time. Of particular interest is the possibility of measuring cell or bacterial growth. High precision measurements of the mass of living cells or small multi-cellular organisms such as bacteria have not been possible previously.
Given the mass resolution of current SMR's cell mass measurements may be accomplished with resolution approximately 1% of the mass of a typical bacterium, which is sufficient to measure mass change due to growth and/or mitosis. With such resolution it is possible to detect bacterial growth by measuring change in mass over time, and potentially even more importantly to measure bacterial response to changes in the chemical or environmental properties of the bacteria's liquid environment. Such measurements would have applicability in drug resistance/susceptibility studies, and general environmental toxicity studies. More than 100,000 deaths per year in the US result from bacterial infections, behind only cancer and heart disease. Moreover the rising resistance of bacteria to existing antibiotics coupled with the difficulty in developing new antibiotics is negatively impacting effective treatment of bacterial infections. One of the leading roadblocks to analysis of bacterial resistance to existing drugs and the effectiveness of new drugs is the slowness of existing bacterial assay techniques, such as disk diffusion/dilution assays, which commonly take days to produce results. Therefore it is the object of this invention to provide faster, accurate bacterial assays based on the application of SMR's.